APOE genotype and sex modulate Alzheimer’s disease pathology in aged EFAD transgenic mice

Increasing evidence supports that age, APOE and sex interact to modulate Alzheimer’s disease (AD) risk, however the underlying pathways are unclear. One way that AD risk factors may modulate cognition is by impacting amyloid beta (Aβ) accumulation as plaques, and/or neuroinflammation Therefore, the goal of the present study was to evaluate the extent to which age, APOE and sex modulate Aβ pathology, neuroinflammation and behavior in vivo. To achieve this goal, we utilized the EFAD mice, which express human APOE3 or APOE4 and have five familial AD mutations (FAD) that result in Aβ42 overproduction. We assessed Aβ levels, reactive glia and Morris water maze performance in 6-, 10-, 14-, and 18-month-old EFAD mice. Female APOE4 mice had the highest Aβ deposition, fibrillar amyloid deposits and neuroinflammation as well as earlier behavior deficits. Interestingly, we found that female APOE3 mice and male APOE4 mice had similar levels of pathology. Collectively our data support that the combination of APOE4 and female sex is the most detrimental combination for AD, and that at older ages, female sex may be equivalent to APOE4 genotype.


Introduction
Alzheimer's disease (AD) is a progressive neurodegenerative disease associated with deficits in the cognitive system (Warren et al., 2023).Important risk factors for sporadic AD include age, and sex (Riedel et al., 2016).Age is the greatest overall AD risk factor, with 1 in 9 people over the age of 65 diagnosed with AD (Rajan et al., 2021).APOE, the gene encoding apolipoprotein Balu et al. 10.3389/fnagi.2023.1279343Frontiers in Aging Neuroscience 02 frontiersin.org One way that AD risk factors may modulate cognition is by impacting amyloid beta (Aβ) accumulation as plaques, and/or neuroinflammation.In fact, APOE4 is associated with greater levels of Aβ (Kok et al., 2009) and higher neuroinflammation (Reale et al., 2012;Ringman et al., 2012;Tzioras et al., 2019;Friedberg et al., 2020) compared to non-carriers.In addition, older women generally have higher levels of amyloid pathology compared to men (Oveisgharan et al., 2018) and dysregulated neuroinflammatory responses (Klein and Flanagan, 2016).These human data are recapitulated in vivo in young mice, as APOE4 is associated with increased Aβ deposition and neuroinflammation, compared to APOE3 mice (Youmans et al., 2012b;Rodriguez et al., 2014;Stephen et al., 2019Stephen et al., , 2022)).Therefore, evaluating the extent that APOE and sex impact pathology at older ages is important for advancing our understanding of how each risk factor contributes to AD pathogenesis across the lifespan.
The goal of our study was to understand the extent that APOE and sex interact with age to impact Aβ levels, neuroinflammation, neuronal density and behavior in vivo.To this end, we used EFAD mice as they express human APOE3 (E3FAD) or APOE4 (E4FAD) and overproduce Aβ42.Pathology and behavior were assessed in male and female EFAD mice at 6-, 10-, 14-, and 18-months-of-age using biochemistry, immunohistochemistry, and the Morris water maze test.

Animals
All experiments follow the University of Illinois at Chicago Animal Care Committee protocols.EFAD mice express five familial AD (FAD) mutations and human APOE.Four groups of EFAD (5xFAD +/− /human APOE +/+ ) mice were used: male E3FAD, female E3FAD, male E4FAD, and female E4FAD mice as described previously (Youmans et al., 2012b).Mice from each of the four groups (male and female, E3FAD and E4FAD mice) were enrolled by age (i.e., 6, 10, 14, or 18 months euthanized together) as the breeding schedule permitted.Mice were ear-tagged during genotyping and investigators conducting experiments and data analysis were blinded for APOE genotype, sex, and age.Two cohorts of mice were used in this study (n = 13-25), one was used for biochemical and immunohistochemical analysis and the second for behavior analysis using Morris water maze.

Brain tissue harvest and processing
Mice were anesthetized via intraperitoneal injection with ketamine (100 mg/kg) and xylazine (5 mg/kg) and perfused transcardially with ~40 mL phosphate buffered saline with protease inhibitor cocktail.Then, the brains were removed and dissected at the midline to produce two hemi-brains, one each for immunohistochemical and biochemical analysis (Youmans et al., 2012b;Valencia-Olvera et al., 2023).The hemi for immuno histochemical analysis was drop-fixed in 4% paraformaldehyde for 24 h and then transferred to phosphate buffered saline containing 0.01% sodium azide until ready to section on a sliding microtome.Cortex was dissected from the hemi-brain for biochemical analysis, flash-frozen in liquid nitrogen and then stored at −80°C.

Immunohistochemical analysis
Serial sagittal brain sections (35 μm thick, 280 μm apart, ~0.24 mm -3.44 mm lateral) from EFAD mice were immunostained for Aβ deposition, astrogliosis or microgliosis (Youmans et al., 2012b;Rodriguez et al., 2014;Valencia-Olvera et al., 2023) and stained for fibrillar amyloid deposition via Thio-S (see full list of antibodies and reagents used in this study in Supplementary Table S1).The stained sections were imaged at 10X magnification with a Zeiss Fluorescence microscope and analyzed for cortical area covered by MOAB-2, Thio-S, GFAP and Iba-1 in the cortex using ImageJ by investigators blinded to age, APOE genotype, and sex.Serial sagittal sections (three sections between ~0.72-and 1.80-mm lateral) from EFAD mice were stained for NeuN and neuron numbers quantification in layer 5 of the somatosensory cortex, was performed by a design-based stereology system (Stereo Investigator 9, MBF Bioscience, Williston, VT, USA) using an optical fractionator applying the N v × V ref method (West, 2002;Schmitz and Hof, 2007).

Biochemical analysis
Cortices were homogenized in 70% formic acid at 1 mL/150 mg brain tissue and mixed by end-over-end rotation at room temperature for 2 h with vortexing.Samples were then centrifuged (100,000 × g, 1 h at 4°C), and the formic acid-soluble fraction was neutralized (with 20 volumes of 1 M Tris base), aliquoted, and frozen at −80°C (Youmans et al., 2011).Total protein in the formic acid soluble extracts was quantified using the Bradford assay and formic acid-soluble Aβ42 was measured by ELISA following the manufacturer's instructions (Youmans et al., 2011(Youmans et al., , 2012a,b),b).Cortical drebrin levels were measured using Western blotting as described previously (Valencia-Olvera et al., 2023).

Behavioral test: Morris water maze
All behavioral data were recorded and analyzed with ANY-maze software (Stoelting Co, Wood Dale, IL, USA).In the week prior to sacrifice, mouse behavior was tested using a modified Morris water maze protocol with acquisition trials consisting of 4 × 1 min trials/day for 5 consecutive days with latency to the platform recorded for each trial.A single probe trial was run on day 6 with the platform removed, and the readouts included latency to platform area, platform area crosses and latency to target quadrant (Liu et al., 2015;Thomas et al., 2017;Valencia-Olvera et al., 2023).

Data and statistical analysis
Supplementary File S1 is a word file containing Supplementary Table S1 and Supplementary Figures S1-S6.Supplementary File S2 is an excel file containing all raw data and statistical analysis tables including number of mice used for each experiment.Morris water maze acquisition phase data was analyzed by using repeated measure univariate general linear model for within subjects' effects with the independent variables day, age, APOE and sex in SPSS (IBM SPSS Statistics for Macintosh, Version 29.0.1.1).All other statistical 10.3389/fnagi.2023.1279343Frontiers in Aging Neuroscience 03 frontiersin.organalyses were conducted using univariate general linear models for between subjects' effects with the independent variables (age, APOE genotype, sex) and their interactions, followed by Bonferroni's post hoc tests in SPSS.For all statistical tests, p < 0.05 was considered significant.All data are presented as scatter plots with the mean and standard error of mean (SEM).The n size range is indicated in Figure legends.Outliers were excluded using the ROUT method (Q = 5%).All data, detailed n sizes and statistical analysis are provided in Supplementary File S2.

Results
The goal of our study was to determine the extent to which age, APOE and sex modulates Aβ pathology, neuroinflammation and behavior in vivo.To this end, we used EFAD mice that overproduce Aβ42 (via 5xFAD mutations) and express human APOE3 (E3FAD) or APOE4 (E4FAD).EFAD mice were developed to identify how APOE modulates progression of AD-relevant pathology and behavior (Youmans et al., 2012b).Previous studies in EFAD mice have demonstrated that at younger ages (≤ 6 months), Aβ levels, reactive microglia and astrocytes are higher with APOE4 compared to APOE3 (Youmans et al., 2012b;Stephen et al., 2019Stephen et al., , 2022)), and that there are behavioral deficits in female APOE4 mice (Liu et al., 2015).Therefore, we determined the extent that APOE, age and sex impact Aβ pathology, gliosis, and behavior in young (6 and 10 months), middleaged (14 months) and old (18 months) EFAD mice.

APOE and sex impact Aβ pathology: E4FAD females > E4FAD males = E3FAD females > E3FAD
Extracellular Aβ in fibrillar amyloid deposits are used to diagnose AD and are a current therapeutic target (Zhang Y. et al., 2023).Although APOE4 is associated with higher amyloid deposits in human and in vivo, the extent that APOE and sex modulate extracellular Aβ with age is unclear.Therefore, we initially evaluated cortical extracellular Aβ levels (MOAB-2 immunostaining for Aβ: Figure 1A) and fibrillar amyloid deposits (Thio-S staining: Supplementary Figure S1A) in male and female, E3FAD and E4FAD mice.We found that age interacts with APOE to modulate Aβ and fibrillar amyloid deposition (Figures 1B,C).This interaction was due to greater levels of Aβ at 10 and 18 months and amyloid deposits at 10, 14, and 18 months of age, but not at 6 months, in E4FAD mice compared to E3FAD mice.In general, amyloid deposition increased at every age (18 > 14 > 10 > 6 months) in E4FAD mice, whereas levels plateaued at ~10-14 months of age in E3FAD mice (Figures 1B,C).There was also an interaction between age and sex in modulating Aβ deposition, with greater levels in females compared to males at older ages (10, 14, and 18 months) but not at younger ages (6 months).For fibrillar amyloid deposition there was an APOE by sex interaction as levels were in the order E4FAD females > E4FAD males = E3FAD females > E3FAD males (Figure 1C), however there was no age x sex interaction (Supplementary Figure S1B).We also evaluated the levels of formic acid soluble Aβ42 in the cortex (Supplementary Figure S2).In general, the pattern of insoluble Aβ42 levels appears like extracellular Aβ deposition, however the levels were more varied.Thus, at the statistical level formic acid Aβ42 levels were higher with APOE4 compared to APOE3 and in females vs. males at older ages (10-18 months).
Collectively these data support that female sex and APOE have a strong effect on extracellular Aβ deposition.That interaction results in highest fibrillar amyloid deposits in female APOE4 carriers, equivalent levels in female APOE3 and male APOE4 carriers, and lowest levels in male APOE3 carriers at older ages.

APOE and sex affect neuroinflammation: E4FAD females > E3FAD females ≥ E4FAD males > E3FAD males
APOE4 is associated with higher inflammatory responses in younger EFAD mice (Vitek et al., 2012;Rodriguez et al., 2014;Stephen et al., 2022) and in humans (Reale et al., 2012;Ringman et al., 2012;Tzioras et al., 2019;Friedberg et al., 2020).Therefore, we next evaluated the extent that APOE, age and sex impacted levels of reactive astrocytes (GFAP: Figure 2A) and microglia (Iba-1: Figure 2C) via immunostaining, in male and female, E3FAD and E4FAD mice.We found that GFAP coverage was higher with APOE4 compared to APOE3, and for Iba-1 there was interaction between APOE and age with higher coverage in E4FAD mice compared to E3FAD mice at 18 months of age with no effect at other ages (there were also no interactions between age and APOE or APOE and sex, Supplementary Figure S3A).As for extracellular Aβ, we found that age interacts with sex to modulate both astrogliosis and microgliosis.This interaction was due to significantly greater GFAP and Iba-1 coverage in the cortex of female mice between 10 and 18 months (Figures 2B,D).Furthermore, both percentage area covered by GFAP and Iba-1 increased at every age (18 > 14 > 10 > 6 months) in female mice, whereas levels plateaued at ~10-14 months of age in male mice (Figures 2B,D).Overall, these results support that neuroinflammation measured as gliosis was significantly impacted by female sex and APOE.

Female E4FAD mice have early behavioral deficits in acquisition phase of Morris water maze
AD is associated with changes in cognitive trajectory in humans (Jack et al., 2015;Rasmussen et al., 2018) and behavioral deficits in FAD mice (Webster et al., 2014;Jankowsky and Zheng, 2017) including EFAD mice (Liu et al., 2015;Marottoli et al., 2017;Thomas et al., 2017).Therefore, we evaluated the effect of age, APOE and sex in EFAD mice using a modified Morris water maze protocol.It is important to note that our protocol did not include a visible platform.Therefore, we cannot discern whether the swim speeds differ among the four cohorts of mice with age, which in turn may affect their performance in the water maze test.In the acquisition phase, we found that there was an interaction among age, APOE, sex and training day.This interaction was due to a few factors, one of which was differences among groups at each age.When comparing training day 1 to 5, all the four groups of mice learned the location of the platform 6 months of age (Figure 3A: 6 M) (i.e., no effect of APOE or APOE and sex impact Aβ pathology: E4FAD females > E4FAD males = E3FAD females > E3FAD.Cortical extracellular Aβ deposits were assessed using histochemical analysis in male and female, E3FAD and E4FAD mice.(A) Brain sections were immunostained for Aβ using MOAB-2 (Red, scale bars: 1000 μm) and the percentage area quantified (B).(C) Brain sections stained with Thio-S for fibrillar amyloid deposits (see Supplementary Figure S1 for representative images) were quantified for cortical amyloid burden.Data are expressed as mean ± S.E.M and analyzed by univariate general linear modeling followed by Bonferroni's post-hoc analysis (n = 9-15, # APOE/sex difference within an age group, * vs. previous age within APOE/sex combination p < 0.05).See Supplementary File S2 for detailed n sizes and statistical analysis.Sex impact neuroinflammation: E4FAD females > E3FAD females > E4FAD males > E3FAD males.Reactive astrocytes and microglia were assessed using immunohistochemical analysis in male and female, E3FAD and E4FAD mice.(A) Brain sections were immunostained for astrogliosis using GFAP (green, (Continued) Balu et al. 10.3389/fnagi.2023.1279343Frontiers in Aging Neuroscience 06 frontiersin.orgsex).However, at 10-and 14-months of age, female E4FAD mice did not improve in performance from day 1 to day 5 (Figure 3A: 10 M and 14 M).Furthermore, at 18 months of age, only male E3FAD mice showed a significant latency reduction from day 1 to day 5 (Figure 3A: 18 M).These data supported that female E4FAD mice had impairments in acquisition at an earlier age than other groups.Infact, when the day 5 latencies at 6 months of age were compared to other ages, we found deficits at 10 months in E4FAD female mice, at scale bars: 1000 μm) and the percentage area of cortex covered was quantified (B).(C) Reactive microglia in brain sections were immunostained for using Iba-1 (green, scale bars: 1000 μm) and the percentage area of cortex was quantified (D).Data are expressed as mean ± S.E.M and analyzed by univariate general linear modeling followed by Bonferroni's post-hoc analysis (n = 9-15, # APOE/sex difference within an age group, * vs. previous age within APOE/sex combination p < 0.05).See Supplementary File S2 for detailed n sizes and statistical analysis.
FIGURE 2 (Continued) Balu et al. 10.3389/fnagi.2023.1279343Frontiers in Aging Neuroscience 07 frontiersin.org18 months in E3FAD females and E4FAD males, with no changes in E3FAD males (Supplementary Figure S4A).We also evaluated memory 24 h after the last training day using a single probe trial.We found that most probe measures (latency to platform, latency to target quadrant and platform crosses) were impacted by APOE (Figure 3B).However, age, APOE and sex did not affect time spent in target quadrant during the probe trials (Supplementary Figure S4B).Also, there were no interactions among age, APOE and sex for the probe trial measures (Supplementary Figures S4C-F).Importantly, the latency to platform was independently modulated by APOE due to a greater latency to find the platform area and target quadrant in E4FAD compared to E3FAD mice.We next assessed neuronal density (NeuN) in layer 5 of somatosensory cortex in male and female, E3FAD and E4FAD mice (Supplementary Figure S5A).Similar to the probe trial measures for memory, we found that neuronal density (Supplementary Figure S5B) and Drebrin levels (Supplementary Figure S5C) were impacted by age (lower with age) and APOE (APOE4 > APOE3).Collectively, our data support that E4FAD females demonstrated earlier behavioral deficits and loss of neurons at 6-10 months of age, compared to the other groups.

Aβ deposition
Aβ deposition as plaques is one of the main AD pathologies (Rodrigue et al., 2009;Ruan and Sun, 2023;Suemoto and Leite, 2023).As a well-established AD risk (Kawas et al., 2000;Farfel et al., 2019), age has been proposed to modulate Aβ deposition.However, amyloid deposits occur in about 20% of cognitively normal elderly subjects and therefore may also be modulated by other AD risk factors.APOE has a pronounced impact on Aβ deposition.Aβ levels are higher with APOE4 in human (Villemagne et al., 2011;Chiotis et al., 2015;Jack et al., 2015;Resnick et al., 2015;Mishra et al., 2018;Ba et al., 2019;Baek et al., 2020;Therriault et al., 2020Therriault et al., , 2021;;Cacciaglia et al., 2022) and mouse studies (Youmans et al., 2012b;Moser and Pike, 2017;Montagne et al., 2021).Sex is also thought to impact Aβ deposition.The independent effect of sex in modulating Aβ deposition is conflicting in humans (Jack et al., 2015;Ba et al., 2019;Therriault et al., 2020Therriault et al., , 2021;;Cacciaglia et al., 2022), potentially due to variable hormone levels, disease stage, co-morbidities and other lifestyle factors.However, in FAD mice, females have greater Aβ pathology compared to males (Wang et al., 2003;Li et al., 2016;Dennison et al., 2021;Forner et al., 2021;Marazuela et al., 2022;Sil et al., 2022).Our data agree with in vivo and human studies that the combination of APOE4 and female sex results in highest levels of Aβ deposition compared to other groups (Figure 1; Supplementary Figure S2).Interestingly, our data also demonstrate that Aβ pathology is equivalent in female E3FAD mice and male E4FAD mice.These novel findings raise the possibility that female sex and APOE4 induce a similar impact on Aβ pathology.These findings support that female sex may be an important factor for enrolling in preclinical/clinical trials targeting Aβ (Shi et al., 2022).

Neuroinflammation
Neuroinflammation is also an important AD pathology, often defined as levels of reactive astrocytes and microglia (glia) in the brain (Streit et al., 2004).In general, in AD patients higher levels of reactive glia are thought to occur early in disease progression (Hoozemans et al., 2011), in females compared to males (Duarte-Guterman et al., 2020;Lynch, 2022) and with APOE4 (Reale et al., 2012;Ringman et al., 2012;Tzioras et al., 2019;Friedberg et al., 2020).However, the interaction among age, APOE and sex on reactive glia has yet to be characterized in AD patients, and therefore FAD mouse models are currently being used to address this issue.In vivo studies demonstrate that there is greater neuroinflammation in females compared to males in FAD mice (Hou et al., 2015;Schmid et al., 2019;Guillot-Sestier et al., 2021;Mifflin et al., 2021;Sil et al., 2022).This sex effect is pronounced with APOE4 in young mice (Stephen et al., 2019(Stephen et al., , 2022)).We have extended these findings to older mice and demonstrate that the combination of APOE4 and female sex results in the highest levels of reactive glia with age.In addition, our data revealed that in general, female E3FAD mice have greater neuroinflammation compared to male E4FAD mice.Collectively, our data demonstrates that targeting neuroinflammation may be an attractive therapeutic approach for females AD patients.

Behavior
AD is typically characterized by memory loss, and a decline in overall cognitive function (Rao et al., 2023).Several studies demonstrate that with age, cognitive decline is accelerated in AD patients (Rosselli et al., 2000;Conde-Sala et al., 2013;Zhao et al., 2014;Pan et al., 2021) and FAD mice (Middei et al., 2006;Ferguson et al., 2013;Webster et al., 2013;Olesen et al., 2016;Forner et al., 2021;Locci et al., 2021).Among AD patients, cognitive decline is pronounced in APOE4, compared to APOE3 carriers (Emrani et al., 2020;Gharbi-Meliani et al., 2021;Qian et al., 2021Qian et al., , 2023;;Polsinelli et al., 2023b).This effect is mirrored in FAD mice in the presence of APOE4, compared to APOE3 in young (Thomas et al., 2017) and old (Montagne et al., 2021) mice.In addition, sex impacts cognitive decline in AD patients and memory deficits in mice.In general, women display poorer cognitive profiles compared to men at the same stage of AD (Subramaniapillai et al., 2021) and is mirrored in female mice compared to male FAD mice (Poon et al., 2023), also reviewed in Li and Singh (2014) and Laws et al. (2016).In this study, we evaluated the combined effect of age, APOE and sex in Morris water maze performance and found that female E4FAD mice had earlier behavioral deficits during acquisition phase of Morris water maze compared to other groups (Figure 3A).Consistent with other readouts, APOE3 female and APOE4 male had similar behavioral deficits with age.

Limitations
There are some limitations due to the nature of our design.One issue is that we are limited in the extent that we can conclude APOE, sex and age impacted all aspects of AD pathology and behavior.Future studies could incorporate more detailed evaluation of Aβ production, clearance, and degradation pathways, the full neuroinflammatory phenotype, other AD-prevalent pathologies such as vascular function and metabolism, neuronal function, and a full battery of behavioral tests in the cognitive domain.Furthermore, it is important to validate our findings in other models that incorporate human APOE and human APP.For example, human APP knock in mice have been crossed with APOE targeted replacement mice, and data supports greater behavioral deficits and Aβ pathology with APOE4 (Holden et al., 2022).In addition, understanding the effect APOE2 genotype which is protective for AD risk is important, with the caveat that these mice have Type III hyperlipoproteinemia (Sullivan et al., 1998).Our study raises an important discussion of the possible underlying mechanisms of how APOE4 and female sex impact AD pathology and cognition.For example, structural differences between apoE isoforms may modulate Aβ levels, glial reactivity, and neuron function [reviewed in Hunsberger et al. (2019), Martinez-Martinez et al. (2020), Raulin et al. (2022), Yang et al. (2023), andZhang L. et al. (2023)].As for female sex, the increase in AD risk/pathology has been attributed to loss of sex hormones with age particularly at menopause in humans (Rocca et al., 2011;Levine et al., 2016;Jett et al., 2022;Mishra et al., 2022), which is recapitulated in ovariectomized mice (Ge et al., 2020;Sanchez et al., 2023).Although mice do not have the typical menopausal symptoms as humans, there are age-dependent changes in hormonal levels, which could impact AD-pathology and cognition in mice.Indeed, we observed that the number of mice in proestrus/estrus decrease with age in female EFAD mice (Supplementary Figure S6).In addition, there may be X chromosomemediated modulation of AD risk (Guo et al., 2022) may partly explain the sex-biased differences in AD.Furthermore, our study has also raised questions for future research including: What aspect of AD pathology is proximal to behavior changes in EFAD mice?What is the effect of APOE3/4 genotype on AD pathology and the interaction with sex?Does the impact of sex and APOE on pathology modify responses to therapeutic treatments?

Conclusion
Our data support that the combination of female sex and APOE4 result in the greatest levels of Aβ pathology, neuroinflammation and behavioral impairments.In addition, we propose that the effect of female sex is analogous to the presence of APOE4 for AD pathology.Through reviewing previous published in vivo and human data, we believe that this important concept may have been overlooked (Holland et al., 2013;Huynh et al., 2019;Stephen et al., 2019Stephen et al., , 2022;;Cho et al., 2021;Montagne et al., 2021;Palmer et al., 2022).The female sex and APOE4 equivalency could therefore impact the optimal treatment window for therapeutics aimed at preventing AD through targeting Aβ and/or neuroinflammation.

FIGURE 3 Female
FIGURE 3Female E4FAD mice have early behavioral deficits.Learning and memory were assessed via Morris water maze.(A) EFAD mice were trained to locate the location of a platform over 5 days (acquisition phase) and (B) the ability to remember the location of the platform 24 h after the last training day (probe trial).Data are expressed as mean ± S.E.M. Latency to platform during acquisition phase was analyzed by repeated measure general linear modeling followed by Bonferroni's post-hoc analysis (n = 9-15, § day 1 vs. day 5 matched by mouse group color, p < 0.05).Probe trial measures were analyzed by univariate general linear modeling followed by Bonferroni's post-hoc analysis (n = 9-15, # APOE/sex difference within an age group, * vs. previous age within APOE/sex combination p < 0.05).See Supplementary File S2 for detailed n sizes and statistical analysis.